Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

Abstract

Previously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate of Leishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance in L. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by >3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ϳ5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate. L. donovani PCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate of L. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (Sb V ) and trivalent antimonial (Sb III ) drugs was assessed in vitro against promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC 50 ) of Sb V (from 41.2 ؎ 0.6 g/ml to 66.5 ؎ 3.9 g/ml) and Sb III (from 24.0 ؎ 0.3 g/ml to 43.4 ؎ 1.8 g/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon Sb III treatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance in L. donovani clinical isolates, which warrants detailed investigations regarding its mechanism.

Deviating the level of proliferating cell nuclear antigen inTrypanosoma bruceielicits distinct mechanisms for inhibiting proliferation and cell cycle progression

“…40 The phenotype observed after upregulating TbPCNA in T. brucei was in stark contrast to that reported for upregulating it in human cells or in L. donovani. Reduced proliferation in non-induced TbPCNA OE clones was correlated with marginal increases of intra-parasite TbPCNA levels caused by the lax plasmid promoter.…”

“…Similar amino acid insertions were recognized in PCNA homologs from the related kinetoplastid parasite Leishmania donovani. 40 As a member of the sliding clamp family, it is predicted that TbPCNA can assume a toroid structure similar to other PCNA homologs. The homology model for TbPCNA, based on monomer structures of yeast and human PCNAs (PDBs 3K4X and 3VKX respectively), predicted that it had 2 domains bridged by an interdomain-connecting loop (Fig.…”

Leishmanicidal Activity of Isoselenocyanate Derivatives

“…Furthermore, among the molecules associated with Leishmania replication, some of them related to treatment were considered of interest and studied. For example, PCNA had been described to exhibit a significant role in drug response in Leishmania (7,28). On the other hand, among the DNA helicases probably involved in DNA replication, MCM4 (minichromosome maintenance complex 4) had been mentioned as one of the most sensitive to drug, such as heliquinomycin (29), and MCM4 had been found in Leishmania, too (30).…”

“…Moreover, in Leishmania, PCNA colocalizes with MCM4 in the S phase of the cell cycle (30). In 2014, Tandon et al demonstrated that PCNA plays a significant role in drug resistance in Leishmania clinical specimens (28). Therefore, TOP-2, PCNA, and MCM4 are key molecules for Leishmania biology.…”

DNA vaccine against visceral leishmaniasis: a promising approach for prevention and control

“…The parasite is responsible for a spectrum of clinical syndromes, which can, in most extreme cases, move from an asymptomatic infection to a fatal form of VL. The available antileishmanial drugs are toxic, having serious side effects and are associated with numerous relapses, and there is an increasing incidence of drug resistance also . However, resistance in clinical field isolates is not common, for example AmBisome (liposomal amphotericin B formulation) is safest and effective drug against VL in India but same regimens was not reported to be suitable for VL treatment across eastern Africa .…”